CiteULike: NicMag's library [96 articles]

Cortical microtubule contacts position the spindle in C. elegans embryos.

C Kozlowski — 2007-07-02T09:26:51-00:00

Cell, Vol. 129, No. 3. (4 May 2007), pp. 499-510.

Interactions between microtubules and the cell cortex play a critical role in positioning organelles in a variety of biological contexts. Here we used Caenorhabditis elegans as a model system to study how cortex-microtubule interactions position the mitotic spindle in response to polarity cues. Imaging EBP-2::GFP and YFP::alpha-tubulin revealed that microtubules shrink soon after cortical contact, from which we propose that cortical adaptors mediate microtubule depolymerization energy into pulling forces. We also observe association of dynamic microtubules to form astral fibers that persist, despite the catastrophe events of individual microtubules. Computer simulations show that these effects, which are crucially determined by microtubule dynamics, can explain anaphase spindle oscillations and posterior displacement in 3D.

Spindle positioning by cortical pulling forces.

SW Grill — 2008-06-24T11:50:07-00:00

Developmental cell, Vol. 8, No. 4. (April 2005), pp. 461-465.

Proper spatial control of the cell division plane is essential to any developing organism. In most cell types, the relative size of the two daughter cells is determined by the position of the mitotic spindle within the geometry of the mother cell. We review the underlying mechanisms responsible for positioning of the mitotic spindle, both in cases where the spindle is placed in the center of the cell and in cases where the spindle is placed away from the center of the cell. We discuss the idea that cortical pulling forces are sufficient to provide a general mechanism for spindle positioning within symmetrically and asymmetrically dividing cells.

Theory of mitotic spindle oscillations.

SW Grill — 2008-06-24T15:36:32-00:00

Physical review letters, Vol. 94, No. 10. (18 March 2005)

During unequal cell division the mitotic spindle is positioned away from the center of the cell before cell cleavage. In many biological systems this repositioning is accompanied by oscillatory movements of the spindle. We present a theoretical description for mitotic spindle oscillations. We show that the cooperative attachment and detachment of cortical force generators to astral microtubules leads to spontaneous oscillations beyond a critical number of force generators. This mechanism can quantitatively describe the spindle oscillations observed during unequal division of the one cell stage Caenorhabditis elegans embryo.

Lipids trigger changes in the elasticity of the cytoskeleton in plant cells: a cell optical displacement assay for live cell measurements

S Grabski — 2008-06-03T18:12:27-00:00

Journal of Cell Biololgy, Vol. 126, No. 3. (1 August 1994), pp. 713-726.

10.1083/jcb.126.3.713

Micromanipulation of statoliths in gravity-sensing Chara rhizoids by optical tweezers.

G Leitz — 2008-06-03T18:11:08-00:00

Planta, Vol. 197, No. 2. (September 1995), pp. 278-288.

Infrared laser traps (optical tweezers) were used to micromanipulate statoliths in gravity-sensing rhizoids of the green alga Chara vulgaris Vail. We were able to hold and move statoliths with high accuracy and to observe directly the effects of statolith position on cell growth in horizontally positioned rhizoids. The first step in gravitropism, namely the physical action of gravity on statoliths, can be simulated by optical tweezers. The direct laser microirradiation of the rhizoid apex did not cause any visible damage to the cells. Through lateral positioning of statoliths a differential growth of the opposite flank of the cell wall could be induced, corresponding to bending growth in gravitropism. The acropetal displacement of the statolith complex into the extreme apex of the rhizoid caused a temporary decrease in cell growth rate. The rhizoids regained normal growth after remigration of the statoliths to their initial position 10-30 micrometers basal to the rhizoid apex. During basipetal displacement of statoliths, cell growth continued and the statoliths remigrated towards the rhizoid tip after release from the optical trap. The resistance to statolith displacement increased towards the nucleus. The basipetal displacement of the whole complex of statoliths for a long distance (>100 micrometers) caused an increase in cell diameter and a subsequent regaining of normal growth after the statoliths reappeared in the rhizoid apex. We conclude that the statolith displacement interferes with the mechanism of tip growth, i.e. with the transport of Golgi vesicles, either directly by mechanically blocking their flow and/or, indirectly, by disturbing the actomyosin system. In the presence of the actin inhibitor cytochalasin B the optical forces required for acropetal and basipetal displacement of statoliths were significantly reduced to a similar level. The lateral displacement of statoliths was not changed by cytochalasin B. The results indicate: (i) the viscous resistance to optical displacement of statoliths depend mainly on actin, (ii) the lateral displacement of statoliths is not impeded by actin filaments, (iii) the axially directed actin-mediated forces against optical displacement of statoliths (for a distance of 10 micrometers) are stronger in the basipetal than in the acropetal direction, (iv) the forces acting on single statoliths by axially oriented actin filaments are estimated to be in the range of 11-110 pN for acropetal and of 18-180 pN for basipetal statolith displacements.

Micromanipulation of chloroplasts using optical tweezers.

S Bayoudh — 2008-06-03T18:10:41-00:00

Journal of microscopy, Vol. 203, No. Pt 2. (August 2001), pp. 214-222.

This paper describes experiments using optical tweezers to probe chloroplast arrangement, shape and consistency in cells of living leaf tissue and in suspension. Dual optical tweezers provided two-point contact on a single chloroplast or two-point contact on two adhered chloroplasts for manipulation in suspension. Alternatively, a microstirrer consisting of a birefringent particle trapped in an elliptically polarized laser trap was used to induce motion and tumbling of a selected chloroplast suspended in a solution. We demonstrate that displacement of chloroplasts inside the cell is extremely difficult, presumably due to chloroplast adhesion to the cytoskeleton and connections between organelles. The study also confirms that the chloroplasts are very thin and extremely cup-shaped with a concave inner surface and a convex outer surface.

Impairment of cytoskeleton-dependent vesicle and organelle translocation in green algae: combined use of a microfocused infrared laser as microbeam and optical tweezers.

A Holzinger — 2008-06-03T18:10:01-00:00

Journal of microscopy, Vol. 208, No. Pt 2. (November 2002), pp. 77-83.

A Nd-YAG laser at 1064 nm is used as optical tweezers to move intracellular objects and a laser microbeam to cause impairment of cytoskeleton tracks and influence intracellular motions in desmidiaceaen green algae. Naturally occurring migrations of large nuclei are inhibited in Micrasterias denticulata and Pleurenterium tumidum when the responsible microtubules are targeted with a laser microbeam generating 180 mW power in the focal plane. Impairment of the microtubule tracks appears to be irreversible, as the nucleus cannot pass the former irradiated area in Pleurenterium or remains abnormally dislocated in Micrasterias. The actin filament-dependent movement of secretory vesicles and smaller particles can be manipulated by the same IR-laser at 90 mW when functioning as optical tweezers. In Closterium lunula particles are displaced from their cytoplasmic tracks for up to 10 micro m but return to their tracks immediately after removing the light pressure gained by the optical tweezers. The cytoplasmic tracks consist of actin filament cables running parallel to the longitudinal axis of Closterium cells as depicted by Alexa phalloidin staining and confocal laser scanning microscopy. Dynamics and extensibility of the cytoplasmic strands connecting particles to the tracks are also demonstrated in the area of large vacuoles which are surrounded by actin filament bundles. In Micrasterias trapping of secretory vesicles by the optical tweezers causes irreversible malformations of the cell shape. The vesicle accumulation itself dissipates within 30 s after removing the optical tweezers, also indicating reversibility of the effects induced, in the case of actin filament-mediated processes.

Positioning of nuclei in Arabidopsis root hairs: an actin-regulated process of tip growth.

T Ketelaar — 2008-06-03T18:09:25-00:00

The Plant cell, Vol. 14, No. 11. (November 2002), pp. 2941-2955.

In growing Arabidopsis root hairs, the nucleus locates at a fixed distance from the apex, migrates to a random position during growth arrest, and moves from branch to branch in a mutant with branched hairs. Consistently, an artificial increase of the distance between the nucleus and the apex, achieved by entrapment of the nucleus in a laser beam, stops cell growth. Drug studies show that microtubules are not involved in the positioning of the nucleus but that subapical fine F-actin between the nucleus and the hair apex is required to maintain the nuclear position with respect to the growing apex. Injection of an antibody against plant villin, an actin filament-bundling protein, leads to actin filament unbundling and movement of the nucleus closer to the apex. Thus, the bundled actin at the tip side of the nucleus prevents the nucleus from approaching the apex. In addition, we show that the basipetal movement of the nucleus at root hair growth arrest requires protein synthesis and a functional actin cytoskeleton in the root hair tube.

Femtosecond laser nanoaxotomy lab-on-a-chip for in vivo nerve regeneration studies

Samuel Guo — 2008-05-31T02:04:43-00:00

Nature Methods, Vol. 5, No. 6. (13 April 2008), pp. 531-533.

Two-photon fluorescence absorption and emission spectra of dyes relevant for cell imaging

F Bestvater — 2008-05-27T12:10:47-00:00

Journal of Microscopy, Vol. 208, No. 2. (2002), pp. 108-115.

Summary Two-photon absorption and emission spectra for fluorophores relevant in cell imaging were measured using a 45 fs Ti:sapphire laser, a continuously tuneable optical parametric amplifier for the excitation range 580-1150 nm and an optical multichannel analyser. The measurements included DNA stains, fluorescent dyes coupled to antibodies as well as organelle trackers, e.g. Alexa and Bodipy dyes, Cy2, Cy3, DAPI, Hoechst 33342, propidium iodide, FITC and rhodamine. In accordance with the two-photon excitation theory, the majority of the investigated fluorochromes did not reveal significant discrepancies between the two-photon and the one-photon emission spectra. However, a blue-shift of the absorption maxima ranging from a few nanometres up to considerably differing courses of the spectrum was found for most fluorochromes. The potential of non-linear laser scanning fluorescence microscopy is demonstrated here by visualizing multiple intracellular structures in living cells. Combined with 3D reconstruction techniques, this approach gives a deeper insight into the spatial relationships of subcellular organelles.

Pulse energy dependence of subcellular dissection by femtosecond laser pulses.

A Heisterkamp — 2008-05-26T17:27:57-00:00

Optics Express, Vol. 13, No. 10. (16 May 2005), pp. 3690-3696.

Precise dissection of cells with ultrashort laser pulses requires a clear understanding of how the onset and extent of ablation (i.e., the removal of material) depends on pulse energy. We carried out a systematic study of the energy dependence of the plasma-mediated ablation of fluorescently-labeled subcellular structures in the cytoskeleton and nuclei of fixed endothelial cells using femtosecond, near-infrared laser pulses focused through a high-numerical aperture objective lens (1.4 NA). We find that the energy threshold for photobleaching lies between 0.9 and 1.7 nJ. By comparing the changes in fluorescence with the actual material loss determined by electron microscopy, we find that the threshold for true material ablation is about 20% higher than the photobleaching threshold. This information makes it possible to use the fluorescence to determine the onset of true material ablation without resorting to electron microscopy. We confirm the precision of this technique by severing a single microtubule without disrupting the neighboring microtubules, less than 1 micrometer away.

Optical trapping inside living organisms

PM Hansen — 2007-06-24T19:55:05-00:00

Proc SPIE Int Soc Opt Eng, Vol. 5930, pp. 1-9.

We use optical tweezers to investigate processes happening inside living cells. In a previous study,1 we trapped naturally occurring lipid granules inside living yeast cells, and used them to probe the viscoelastic properties of the cytoplasm. However, we prefer to use probes which can be specifically attached to various organelles within the living cells in order to optically quantify the forces acting on these organelles. Therefore, we have chosen to use nanometer sized gold beads as probes. These gold beads can be conjugated and attached chemically to the organelles of interest. Only Rayleigh metallic particles can be optically trapped2 and for these it is the case that the larger the beads, the larger the forces which can be exerted and thus measured using optical tweezers.3 The gold nanoparticles are injected into the cytoplasm using micropipettes. The very rigid cell wall of the S. pombe yeast cells poses a serious obstacle to this injection. In order to be able to punch a hole in the cell, first, the cells have to be turned into protoplasts, where only a lipid bilayer separates the cytoplasm from the surrounding media. We show how to perform micropipette delivery into the protoplasts and also how the protoplasts can be ablated using the trapping laserlight. Finally, we demonstrate that we can transform the protoplasts back to normal yeast cells. Art. No.: 593003 Sponsors: SPIE _ The International Society for Optical Engineering

Viscoelastic Retraction of Single Living Stress Fibers and Its Impact on Cell Shape, Cytoskeletal Organization, and Extracellular Matrix Mechanics

Sanjay Kumar — 2008-05-16T11:04:54-00:00

Biophysical Journal, Vol. 90, No. 10. (15 May 2006), pp. 3762-3773.

Cells change their form and function by assembling actin stress fibers at their base and exerting traction forces on their extracellular matrix (ECM) adhesions. Individual stress fibers are thought to be actively tensed by the action of actomyosin motors and to function as elastic cables that structurally reinforce the basal portion of the cytoskeleton; however, these principles have not been directly tested in living cells, and their significance for overall cell shape control is poorly understood. Here we combine a laser nanoscissor, traction force microscopy, and fluorescence photobleaching methods to confirm that stress fibers in living cells behave as viscoelastic cables that are tensed through the action of actomyosin motors, to quantify their retraction kinetics in situ, and to explore their contribution to overall mechanical stability of the cell and interconnected ECM. These studies reveal that viscoelastic recoil of individual stress fibers after laser severing is partially slowed by inhibition of Rho-associated kinase and virtually abolished by direct inhibition of myosin light chain kinase. Importantly, cells cultured on stiff ECM substrates can tolerate disruption of multiple stress fibers with negligible overall change in cell shape, whereas disruption of a single stress fiber in cells anchored to compliant ECM substrates compromises the entire cellular force balance, induces cytoskeletal rearrangements, and produces ECM retraction many microns away from the site of incision; this results in large-scale changes of cell shape (> 5% elongation). In addition to revealing fundamental insight into the mechanical properties and cell shape contributions of individual stress fibers and confirming that the ECM is effectively a physical extension of the cell and cytoskeleton, the technologies described here offer a novel approach to spatially map the cytoskeletal mechanics of living cells on the nanoscale. 10.1529/biophysj.105.071506

Neurosurgery: Functional regeneration after laser axotomy

Mehmet Yanik — 2004-12-16T13:06:15-00:00

Nature, Vol. 432, No. 7019. (16 December 2004), pp. 822-822.

Visualizing Spatiotemporal Dynamics of Multicellular Cell-Cycle Progression

Asako Sawano — 2008-02-08T16:32:00-00:00

Cell, Vol. 132, No. 3. (8 February 2008), pp. 487-498.

Summary The cell-cycle transition from G1 to S phase has been difficult to visualize. We have harnessed antiphase oscillating proteins that mark cell-cycle transitions in order to develop genetically encoded fluorescent probes for this purpose. These probes effectively label individual G1 phase nuclei red and those in S/G2/M phases green. We were able to generate cultured cells and transgenic mice constitutively expressing the cell-cycle probes, in which every cell nucleus exhibits either red or green fluorescence. We performed time-lapse imaging to explore the spatiotemporal patterns of cell-cycle dynamics during the epithelial-mesenchymal transition of cultured cells, the migration and differentiation of neural progenitors in brain slices, and the development of tumors across blood vessels in live mice. These mice and cell lines will serve as model systems permitting unprecedented spatial and temporal resolution to help us better understand how the cell cycle is coordinated with various biological events.

The Dam1 kinetochore ring complex moves processively on depolymerizing microtubule ends

Stefan Westermann — 2006-01-16T03:57:45-00:00

Nature (15 January 2006)

Nuclear congression is driven by cytoplasmic microtubule plus end interactions in S. cerevisiae

Jeffrey Molk — 2008-04-29T08:27:35-00:00

J. Cell Biol., Vol. 172, No. 1. (3 January 2006), pp. 27-39.

Nuclear movement before karyogamy in eukaryotes is known as pronuclear migration or as nuclear congression in Saccharomyces cerevisiae. In this study, S. cerevisiae is used as a model system to study microtubule (MT)-dependent nuclear movements during mating. We find that nuclear congression occurs through the interaction of MT plus ends rather than sliding and extensive MT overlap. Furthermore, the orientation and attachment of MTs to the shmoo tip before cell wall breakdown is not required for nuclear congression. The MT plus end-binding proteins Kar3p, a class 14 COOH-terminal kinesin, and Bik1p, the CLIP-170 orthologue, localize to plus ends in the shmoo tip and initiate MT interactions and depolymerization after cell wall breakdown. These data support a model in which nuclear congression in budding yeast occurs by plus end MT capture and depolymerization, generating forces sufficient to move nuclei through the cytoplasm. This is the first evidence that MT plus end interactions from oppositely oriented organizing centers can provide the force for organelle transport in vivo. 10.1083/jcb.200510032

Anaphase Inactivation of the Spindle Checkpoint

William Palframan — 2008-04-29T08:26:10-00:00

Science, Vol. 313, No. 5787. (4 August 2006), pp. 680-684.

The spindle checkpoint delays cell cycle progression until microtubules attach each pair of sister chromosomes to opposite poles of the mitotic spindle. Following sister chromatid separation, however, the checkpoint ignores chromosomes whose kinetochores are attached to only one spindle pole, a state that activates the checkpoint prior to metaphase. We demonstrate that, in budding yeast, mutual inhibition between the anaphase-promoting complex (APC) and Mps1, an essential component of the checkpoint, leads to sustained inactivation of the spindle checkpoint. Mps1 protein abundance decreases in anaphase, and Mps1 is a target of the APC. Furthermore, expression of Mps1 in anaphase, or repression of the APC in anaphase, reactivates the spindle checkpoint. This APC-Mps1 feedback circuit allows cells to irreversibly inactivate the checkpoint during anaphase. 10.1126/science.1127205

Yeast kinesin-8 depolymerizes microtubules in a length-dependent manner

Vladimir Varga — 2006-09-02T02:45:44-00:00

Nature Cell Biology, Vol. 8, No. 9. (13 August 2006), pp. 957-962.

Kinetochore microtubule dynamics and attachment stability are regulated by Hec1.

JG Deluca — 2007-07-02T10:04:59-00:00

Cell, Vol. 127, No. 5. (1 December 2006), pp. 969-982.

Mitotic cells face the challenging tasks of linking kinetochores to growing and shortening microtubules and actively regulating these dynamic attachments to produce accurate chromosome segregation. We report here that Ndc80/Hec1 functions in regulating kinetochore microtubule plus-end dynamics and attachment stability. Microinjection of an antibody to the N terminus of Hec1 suppresses both microtubule detachment and microtubule plus-end polymerization and depolymerization at kinetochores of PtK1 cells. Centromeres become hyperstretched, kinetochore fibers shorten from spindle poles, kinetochore microtubule attachment errors increase, and chromosomes severely mis-segregate. The N terminus of Hec1 is phosphorylated by Aurora B kinase in vitro, and cells expressing N-terminal nonphosphorylatable mutants of Hec1 exhibit an increase in merotelic attachments, hyperstretching of centromeres, and errors in chromosome segregation. These findings reveal a key role for the Hec1 N terminus in controlling dynamic behavior of kinetochore microtubules.

The Schizosaccharomyces pombe EB1 homolog Mal3p binds and stabilizes the microtubule lattice seam.

L Sandblad — 2007-07-02T09:55:41-00:00

Cell, Vol. 127, No. 7. (29 December 2006), pp. 1415-1424.

End binding 1 (EB1) proteins are highly conserved regulators of microtubule dynamics. Using electron microscopy (EM) and high-resolution surface shadowing we have studied the microtubule-binding properties of the fission yeast EB1 homolog Mal3p. This allowed for a direct visualization of Mal3p bound on the surface of microtubules. Mal3p particles usually formed a single line on each microtubule along just one of the multiple grooves that are formed by adjacent protofilaments. We provide structural data showing that the alignment of Mal3p molecules coincides with the microtubule lattice seam as well as data suggesting that Mal3p not only binds but also stabilizes this seam. Accordingly, Mal3p stabilizes microtubules through a specific interaction with what is potentially the weakest part of the microtubule in a way not previously demonstrated. Our findings further suggest that microtubules exhibit two distinct reaction platforms on their surface that can independently interact with target structures such as microtubule-associated proteins, motors, kinetochores, or membranes.

Organization of Interphase Microtubules in Fission Yeast Analyzed by Electron Tomography

Johanna Höög — 2008-04-29T08:17:13-00:00

Developmental Cell, Vol. 12, No. 3. (March 2007), pp. 349-361.

Summary Polarized cells, such as neuronal, epithelial, and fungal cells, all display a specialized organization of their microtubules (MTs). The interphase MT cytoskeleton of the rod-shaped fission yeast, Schizosaccharomyces pombe, has been extensively described by fluorescence microscopy. Here, we describe a large-scale, electron tomography investigation of S. pombe, including a 3D reconstruction of a complete eukaryotic cell volume at sufficient resolution to show both how many MTs there are in a bundle and their detailed architecture. Most cytoplasmic MTs are open at one end and capped at the other, providing evidence about their polarity. Electron-dense bridges between the MTs themselves and between MTs and the nuclear envelope were frequently observed. Finally, we have investigated structure/function relationships between MTs and both mitochondria and vesicles. Our analysis shows that electron tomography of well-preserved cells is ideally suited for describing fine ultrastructural details that were not visible with previous techniques.

Crosslinkers and motors organize dynamic microtubules to form stable bipolar arrays in fission yeast.

ME Janson — 2007-06-10T09:45:22-00:00

Cell, Vol. 128, No. 2. (26 January 2007), pp. 357-368.

Microtubule (MT) nucleation not only occurs from centrosomes, but also in large part from dispersed nucleation sites. The subsequent sorting of short MTs into networks like the mitotic spindle requires molecular motors that laterally slide overlapping MTs and bundling proteins that statically connect MTs. How bundling proteins interfere with MT sliding is unclear. In bipolar MT bundles in fission yeast, we found that the bundler ase1p localized all along the length of antiparallel MTs, whereas the motor klp2p (kinesin-14) accumulated only at MT plus ends. Consequently, sliding forces could only overcome resistant bundling forces for short, newly nucleated MTs, which were transported to their correct position within bundles. Ase1p thus regulated sliding forces based on polarity and overlap length, and computer simulations showed these mechanisms to be sufficient to generate stable bipolar bundles. By combining motor and bundling proteins, cells can thus dynamically organize stable regions of overlap between cytoskeletal filaments.

Architecture of the Dam1 kinetochore ring complex and implications for microtubule-driven assembly and force-coupling mechanisms

Hong Wang — 2007-08-04T12:28:16-00:00

Nature Structural & Molecular Biology, Vol. 14, No. 8. (22 July 2007), pp. 721-726.

Microtubules offset growth site from the cell centre in fission yeast

Stefania Castagnetti — 2008-04-29T08:00:43-00:00

J Cell Sci, Vol. 120, No. 13. (1 July 2007), pp. 2205-2213.

The design principles that underlie cellular morphogenetic mechanisms are central to understanding the generation of cell form. We have investigated the constraints governing the formation and positioning of new growth zones in the fission yeast cell and have shown that establishment of a new axis of polarity is independent of microtubules and that in the absence of microtubules a new growth zone is activated near the nucleus in the middle of the cell. Activation of a new growth zone can occur at any stage of the cell cycle as long as the nucleus is a sufficient distance away from previously growing ends. The positioning of growth zones is regulated by the polarity marker Tea1 delivered by microtubules; cells with short microtubules locate the growth zone near the region where the microtubules terminate. We propose a model for the activation of new growth zones comprising a long-range laterally inhibitory component and a self-activating positive local component that is delivered to cell ends by Tea1 and the microtubules. The principle of this symmetry-breaking design may also apply to the morphogenesis of other cells. 10.1242/jcs.03464

Kinetochore microtubule interaction during S phase in Saccharomyces cerevisiae

Etsushi Kitamura — 2008-01-04T11:39:14-00:00

Genes Dev., Vol. 21, No. 24. (15 December 2007), pp. 3319-3330.

In the budding yeast Saccharomyces cerevisiae, microtubule-organizing centers called spindle pole bodies (SPBs) are embedded in the nuclear envelope, which remains intact throughout the cell cycle (closed mitosis). Kinetochores are tethered to SPBs by microtubules during most of the cell cycle, including G1 and M phases; however, it has been a topic of debate whether microtubule interaction is constantly maintained or transiently disrupted during chromosome duplication. Here, we show that centromeres are detached from microtubules for 12 min and displaced away from a spindle pole in early S phase. These detachment and displacement events are caused by centromere DNA replication, which results in disassembly of kinetochores. Soon afterward, kinetochores are reassembled, leading to their recapture by microtubules. We also show how kinetochores are subsequently transported poleward by microtubules. Our study gives new insights into kinetochoremicrotubule interaction and kinetochore duplication during S phase in a closed mitosis. 10.1101/gad.449407

Optical tweezers and confocal microscopy for simultaneous three-dimensional manipulation and imaging in concentrated colloidal dispersions

Dirk Vossen — 2008-04-13T15:46:40-00:00

Review of Scientific Instruments, Vol. 75, No. 9. (2004), pp. 2960-2970.

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Wave front engineering for microscopy of living cells

Valentina Emiliani — 2008-04-13T15:44:09-00:00

Optics Express, Vol. 13, No. 5. (7 March 2005), pp. 1395-1405.

A new method to perform simultaneously three dimensional optical sectioning and optical manipulation is presented. The system combines a multi trap optical tweezers with a video microscope to enable axial scanning of living cells while maintaining the trapping configuration at a fixed position. This is achieved compensating the axial movement of the objective by shaping the wave front of the trapping beam with properly diffractive optical elements displayed on a computer controlled spatial light modulator. Our method has been validated in three different experimental configurations. In the first, we decouple the position of a trapping plane from the axial movements of the objective and perform optical sectioning of a circle of beads kept on a fixed plane. In a second experiment, we extend the method to living cell microscopy by showing that mechanical constraints can be applied on the dorsal surface of a cell whilst performing its fluorescence optical sectioning. In the third experiment, we trapped beads in a three dimensional geometry and perform, always through the same objective, an axial scan of the volume delimited by the beads.

Laser induced cell fusion in combination with optical tweezers: the laser cell fusion trap.

RW Steubing — 2008-04-13T15:42:06-00:00

Cytometry, Vol. 12, No. 6. (1991), pp. 505-510.

A single-beam gradient force optical trap was combined with a pulsed UV laser microbeam in order to perform laser induced cell fusion. This combination offers the possibility to selectively fuse two single cells without critical chemical or electrical treatment. The optical trap was created by directing a Nd:YAG laser, at a wavelength of 1.06 microns, into a microscope and focusing the laser beam with a high numerical aperture objective. The UV laser microbeam, produced by a nitrogen-pumped dye laser (366 nm), was collinear with the trapping beam. Once inside the trap, two cells could be fused with several pulses of the UV laser microbeam, attenuated to an energy of approximately 1 microJ/pulse in the object plane. This method of laser induced cell fusion should provide increased selectivity and efficiency in generating viable hybrid cells.

Push-me-pull-you: how microtubules organize the cell interior

Iva Tolić-Norrelykke — 2008-04-12T17:23:33-00:00

European Biophysics Journal, DOI 10.1007/s00249-008-0321-0 (2008)

Abstract Dynamic organization of the cell interior, which is crucial for cell function, largely depends on the microtubule cytoskeleton. Microtubules move and position organelles by pushing, pulling, or sliding. Pushing forces can be generated by microtubule polymerization, whereas pulling typically involves microtubule depolymerization or molecular motors, or both. Sliding between a microtubule and another microtubule, an organelle, or the cell cortex is also powered by molecular motors. Although numerous examples of microtubule-based pushing and pulling in living cells have been observed, it is not clear why different cell types and processes employ different mechanisms. This review introduces a classification of microtubule-based positioning strategies and discusses the efficacy of pushing and pulling. The positioning mechanisms based on microtubule pushing are efficient for movements over small distances, and for centering of organelles in symmetric geometries. Mechanisms based on pulling, on the other hand, are typically more elaborate, but are necessary when the distances to be covered by the organelles are large, and when the geometry is asymmetric and complex. Thus, taking into account cell geometry and the length scale of the movements helps to identify general principles of the intracellular layout based on microtubule forces.

Femtosecond laser disruption of subcellular organelles in a living cell

Wataru Watanabe — 2008-04-12T16:21:14-00:00

Optics Express, Vol. 12, No. 18. (2004), pp. 4203-4213.

Subcellular organelles in living cells were inactivated by tightly focusing femtosecond laser pulses inside the cells. Photodisruption of a mitochondrion in living cells was experimentally confirmed by stacking three-dimensional confocal images and by restaining of organelles. The viability of the cells after femtosecond laser irradiation was ascertained by impermeability of propidium iodide as well as by the presence of cytoplasmic streaming.

Intracellular nanosurgery with near infrared femtosecond laser pulses.

K König — 2008-04-12T16:15:10-00:00

Cellular and Molecular Biology, Vol. 45, No. 2. (March 1999), pp. 195-201.

We report on laser-assisted knocking out of genomic nanometer-sized regions within the nucleus of living cells. The intranuclear nanosurgery was possible by application of highly intense near infrared femtosecond laser pulses. The non-contact laser treatment was performed within a closed sterile cell chamber. The destructive multiphoton effect was based on 10(12) W/cm2 light intensities and limited to a sub-femtoliter focal volume of a high numerical aperture objective. We used the intracellular nanoscalpel for highly precise non-contact dissection of Hoechst-labelled chromosomes within a nucleus of a living Chinese hamster ovary cell. Following laser treatment, the cell remained alive and did not show any signs of membrane perturbation. The use of near infrared pulses provide the possibility of non-invasive intracellular nanoprocessing also within living tissue in depths of more than 100 microns.

Laser nanosurgery of single microtubules reveals location-dependent depolymerization rates.

NM Wakida — 2008-04-12T16:14:00-00:00

Journal of Biomedical Optics, Vol. 12, No. 2. (r 2007)

In this study, 532-nm picosecond and 800-nm femtosecond lasers are used in combination with fluorescently labeled tubulin to further elucidate microtubule depolymerization and the effect lasers may have on the resulting depolymerization. Depolymerization rates of targeted single microtubules are dependent on location with respect to the nucleus. Microtubules located near the nucleus exhibit a significantly faster depolymerization rate when compared to microtubule depolymerization rates near the periphery of the cell. Microtubules cut with the femtosecond laser depolymerize at a slower rate than unirradiated controls (p=0.002), whereas those cut with the picosecond laser depolymerize at the same rate as unirradiated controls (p=0.704). Our results demonstrate the ability of both the picosecond and femtosecond lasers to cut individual microtubules. The differences between the two ablation results are discussed.

Effect of pulse shape on the efficiency of multiphoton process: implications for biological microscopy

CJ Bardeen — 2007-01-22T23:49:51-00:00

Journal of Biomedical Optics, Vol. 4 (July 1999), pp. 362-367.

The effects of spectral shape on two photon fluorescence excitation are investigated experimentally using an acousto- optic pulse shaper to modify femtosecond pulses from a Ti:sapphire laser. By using different spectral window shapes, we find that the measured two photon efficiency can vary by a factor of 2 for differently shaped spectral with the same full width at half maximum. We find that these effects are described well by a simple model assuming transform-limited pulses. The fact that even small changes in the spectral wings can significantly affect the efficiency of nonlinear processes has implications for biological multiphoton imaging, where it may be desirable to minimize sample exposure to radiation and maximize fluorescence or harmonic efficiency.

Multiphoton Fluorescence Microscopy

Enrico Gratton — 2008-03-16T02:05:13-00:00

Methods, Vol. 25, No. 1. (September 2001), pp. 103-110.

Multiphoton fluorescence microscopy has now become a relatively common tool among biophysicists and biologists. The intrinsic sectioning achievable by multiphoton excitation provides a simple means to excite a small volume inside cells and tissues. Multiphoton microscopes have a simplified optical path in the emission side due to the lack of an emission pinhole, which is necessary with normal confocal microscopes. This article illustrates examples in which this advantage in the simplified optics is exploited to achieve a new type of measurements. First, dual-emission wavelength measurements are used to identify regions of different phase domains in giant vesicles and to perform fluctuation experiments at specific locations in the membrane. Second, we show how dual-wavelength measurements are used in conjunction with scanning fluctuation analysis to measure the changes in the geometry of the domains and the incipient formation of gel domains when the temperature of the giant vesicles is gradually lowered.

Three-dimensional laser microsurgery in light-sheet based microscopy (SPIM)

Christoph Engelbrecht — 2008-04-11T09:51:00-00:00

Optics Express, Vol. 15, No. 10. (14 May 2007), pp. 6420-6430.

Advances in the life sciences rely on the ability to observe dynamic processes in live systems and in environments that mimic in-vivo situations. Therefore, new methodological developments have to provide environments that resemble physiologically and clinically relevant conditions as closely as possible. In this work, plasma-induced laser nanosurgery for three-dimensional sample manipulation and sample perturbation is combined with optically sectioning light-sheet based fluorescence microscopy (SPIM) and applied to three-dimensional biological model systems. This means: a) working with a biological system that is not confined to essentially two dimensions like cell cultures on cover glasses, b) gaining intrinsic optical sectioning capabilities by an efficient three-dimensional fluorescence imaging system, and c) using arbitrarily-shaped three-dimensional ablation-patterns by a plasma-induced laser ablation system that prevent damage to surrounding tissues. Spatial levels in our biological applications range from sub-microns during delicate ablation of single microtubules over the confined disruption of cell membranes in an MDCK-cyst to the macroscopic cutting of a millimeter-sized Zebrafish caudal fin with arbitrary three-dimensional ablation patterns. Dynamic processes like laser-induced hemocyte migration can be studied with our SPIM-microscalpel in intact, live embryos.

Reduction of higher-order photobleaching in two-photon excitation microscopy.

PP Mondal — 2008-04-11T09:03:08-00:00

Physical Review. E, Statistical, nonlinear, and soft matter physics, Vol. 75, No. 6 Pt 1. (June 2007)

A theoretical microscopic technique is proposed that may reduce multiphoton interaction in the excitation volume of a two-photon microscope. Since higher-order photobleaching is common in two-photon excitation microscopy, the study of thin samples is limited by increased photobleaching and photodamage. This limitation is elevated by using even coherent state light. The advantage of even coherent state light is that only excitation due to an even number of photons can survive. The very first nonzero even excitation (two-photon) can be isolated from the nearby one- and three-photon excitation. Hence the photobleaching due to one- and three-photon excitation can be eliminated and higher-order processes can be minimized owing to their small molecular cross section.

High-order photobleaching of green fluorescent protein inside live cells in two-photon excitation microscopy.

TS Chen — 2008-04-11T09:02:51-00:00

Biochemical and biophysical research communications, Vol. 291, No. 5. (15 March 2002), pp. 1272-1275.

Combination of green fluorescent protein (GFP) and two-photon excitation fluorescence microscopy (TPE) has been used increasingly to study dynamic biochemical events within living cells, sometimes even in vivo. However, the high photon flux required in TPE may lead to higher-order photobleaching within the focal volume, which would introduce misinterpretation about the fine biochemical events. Here we first studied the high-order photobleaching rate of GFP inside live cells by measuring the dependence of the photobleaching rate on the excitation power. The photobleaching rate under one- and two-photon excitation increased with 1-power and 4-power of the incident intensity, respectively, implying the excitation photons might interact with excited fluorophore molecules and increase the probability of photobleaching. These results suggest that in applications where two-photon imaging of GFP is used to study dynamic molecular process, photobleaching may ruin the imaging results and attention should be paid in interpreting the imaging results.

Photobleaching in two-photon excitation microscopy.

GH Patterson — 2007-05-21T16:40:47-00:00

Biophysical Journal, Vol. 78, No. 4. (April 2000), pp. 2159-2162.

The intensity-squared dependence of two-photon excitation in laser scanning microscopy restricts excitation to the focal plane and leads to decreased photobleaching in thick samples. However, the high photon flux used in these experiments can potentially lead to higher-order photon interactions within the focal volume. The excitation power dependence of the fluorescence intensity and the photobleaching rate of thin fluorescence samples ( approximately 1 microm) were examined under one- and two-photon excitation. As expected, log-log plots of excitation power versus the fluorescence intensity and photobleaching rate for one-photon excitation of fluorescein increased with a slope of approximately 1. A similar plot of the fluorescence intensity versus two-photon excitation power increased with a slope of approximately 2. However, the two-photon photobleaching rate increased with a slope > or =3, indicating the presence of higher-order photon interactions. Similar experiments on Indo-1, NADH, and aminocoumarin produced similar results and suggest that this higher-order photobleaching is common in two-photon excitation microscopy. As a consequence, the use of multi-photon excitation microscopy to study thin samples may be limited by increased photobleaching.

On the fundamental imaging-depth limit in two-photon microscopy.

P Theer — 2007-09-10T19:44:52-00:00

J Opt Soc Am A Opt Image Sci Vis, Vol. 23, No. 12. (December 2006), pp. 3139-3149.

We have analyzed how the maximal imaging depth of two-photon microscopy in scattering samples depends on properties of the sample and the imaging system. We find that the imaging depth increases with increasing numerical aperture and staining inhomogeneity and with decreasing excitation-pulse duration and scattering anisotropy factor, but is ultimately limited by near-surface fluorescence with slight improvements possible using special detection strategies.

Effect of pulse duration on two-photon excited fluorescence and second harmonic generation in nonlinear optical microscopy.

S Tang — 2008-04-11T08:58:16-00:00

Journal of Biomedical Optics, Vol. 11, No. 2. (r 2006)

We have developed a multiphoton microscopy (MPM) system using a 12-fs Ti:sapphire laser with adjustable dispersion precompensation in order to examine the impact of pulse duration on nonlinear optical signals. The efficiencies of two-photon-excited fluorescence (TPEF) and second harmonic generation (SHG) were studied for various pulse durations, measured at the sample, ranging from approximately 400 fs to sub-20 fs. Both TPEF and SHG increased proportionally to the inverse of the pulse duration for the entire tested range. Because of improved signal-to-noise ratio, sub-20-fs pulses were used to enhance MPM imaging depth by approximately 160%, compared to 120-fs pulses, in human skin.

Highly nonlinear photodamage in two-photon fluorescence microscopy.

A Hopt — 2008-04-10T18:25:58-00:00

Biophysical Journal, Vol. 80, No. 4. (April 2001), pp. 2029-2036.

Two-photon fluorescence excitation is being increasingly used in laser scan microscopy due to very low photodamage induced by this technique under normal operation. However, excitation intensity has to be kept low, because nonlinear photodamage sets in when laser power is increased above a certain threshold. We studied this kind of damage in bovine adrenal chromaffin cells, using two different indicators of damage: changes in resting [Ca(2+)] level and the degranulation reaction. In agreement with previous studies, we found that, for both criteria, damage is proportional to the integral (over space and time) of light intensity raised to a power approximately 2.5. Thus, widening the laser pulse shape at constant average intensity both in time and in focal volume is beneficial for avoiding this kind of damage. Both measures, of course, reduce the two-photon fluorescence excitation. However, loss of signal can be compensated by increasing excitation power, such that, at constant damaging potential, signals may be even larger with long pulses and large focal volumes, because the exponent of the power law of damage is higher (mu approximately 2.5) than that of the two-photon signal (mu approximately 2).

Resolution scaling in STED microscopy

Benjamin Harke — 2008-04-10T16:55:05-00:00

Optics Express, Vol. 16, No. 6. (17 March 2008), pp. 4154-4162.

We undertake a comprehensive study of the inverse square root dependence of spatial resolution on the saturation factor in stimulated emission depletion (STED) microscopy and generalize it to account for various focal depletion patterns. We used an experimental platform featuring a high quality depletion pattern which results in operation close to the optimal optical performance. Its superior image brightness and uniform effective resolution < 25 nm are evidenced by imaging both isolated and self-organized convectively assembled fluorescent beads. For relevant saturation values, the generalized square-root law is shown to predict the practical resolution with high accuracy.

Handbook of Biological Confocal Microscopy

J Pawley — 2006-09-06T03:51:58-00:00

(04 August 2006)

This third edition of a classic text in biological microscopy includes detailed descriptions and in-depth comparisons of parts of the microscope itself, digital aspects of data acquisition and properties of fluorescent dyes, the techniques of 3D specimen preparation and the fundamental limitations, and practical complexities of quantitative confocal fluorescence imaging.

Automatic tracking of individual fluorescence particles: application to the study of chromosome dynamics.

D Sage — 2008-04-10T12:49:45-00:00

IEEE transactions on image processing : a publication of the IEEE Signal Processing Society, Vol. 14, No. 9. (September 2005), pp. 1372-1383.

We present a new, robust, computational procedure for tracking fluorescent markers in time-lapse microscopy. The algorithm is optimized for finding the time-trajectory of single particles in very noisy dynamic (two- or three-dimensional) image sequences. It proceeds in three steps. First, the images are aligned to compensate for the movement of the biological structure under investigation. Second, the particle's signature is enhanced by applying a Mexican hat filter, which we show to be the optimal detector of a Gaussian-like spot in 1/omega2 noise. Finally, the optimal trajectory of the particle is extracted by applying a dynamic programming optimization procedure. We have used this software, which is implemented as a Java plug-in for the public-domain ImageJ software, to track the movement of chromosomal loci within nuclei of budding yeast cells. Besides reducing trajectory analysis time by several 100-fold, we achieve high reproducibility and accuracy of tracking. The application of the method to yeast chromatin dynamics reveals different classes of constraints on mobility of telomeres, reflecting differences in nuclear envelope association. The generic nature of the software allows application to a variety of similar biological imaging tasks that require the extraction and quantitation of a moving particle's trajectory.

Dynamics of interphase microtubules in Schizosaccharomyces pombe.

DR Drummond — 2007-11-30T10:41:36-00:00

Current Biology, Vol. 10, No. 13. (29 June 2000), pp. 766-775.

BACKGROUND: Microtubules in interphase Schizosaccharomyces pombe are essential for maintaining the linear growth habit of these cells. The dynamics of assembly and disassembly of these microtubules are so far uncharacterised. RESULTS: Live cell confocal imaging of alpha1 tubulin tagged with enhanced green fluorescent protein revealed longitudinally oriented, dynamically unstable interphase microtubule assemblies (IMAs). The IMAs were uniformly bright along their length apart from a zone of approximately doubly intense fluorescence commonly present close to their centres. The ends of each IMA switched from growth ( approximately 3.0 microm/min) to shrinkage ( approximately 4.5 microm/min) at 1.0 events per minute and from shrinkage to growth at 1.9 events per minute, and the two ends were equivalently dynamic, suggesting equivalent structure. We accordingly propose a symmetrical model for microtubule packing within the IMAs, in which microtubules are plus ends out and overlap close to the equator of the cell. IMAs may contain multiple copies of this motif; if so, then within each IMA end, the microtubule ends must synchronise catastrophe and rescue. When both ends of an IMA lodge in the hemispherical cell ends, the IMAs start to bend under compression and their overall growth rate is inhibited about twofold. Similar microtubule dynamics were observed in cells ranging in size from half to twice normal length. Patterned photobleaching indicated no detectable treadmilling or microtubule sliding during interphase. CONCLUSIONS: The consequence of the mechanisms described is continuous recruitment of microtubule ends to the ends of growing cells, supporting microtubule-based transport into the cell ends and qualitatively accounting for the essential role for microtubules in directing linear cell growth in S. pombe.

Force generation by dynamic microtubules.

M Dogterom — 2007-09-18T15:43:23-00:00

Curr Opin Cell Biol, Vol. 17, No. 1. (February 2005), pp. 67-74.

The assembly and disassembly of microtubules can generate pushing and pulling forces that, together with motor proteins, contribute to the correct positioning of chromosomes, mitotic spindles and nuclei in cells. In vitro experiments combined with modeling have shed light on the intrinsic capability of dynamic microtubules to generate force, and various observations of positioning processes in cells and model systems have shown how pushing and pulling forces are used in different situations. A sophisticated set of microtubule-end-binding proteins is responsible for steering dynamic microtubules toward their cellular target and regulating the pushing and/or pulling forces that are generated once contact is established.

Oscillatory nuclear movement in fission yeast meiotic prophase is driven by astral microtubules, as revealed by continuous observation of chromosomes and microtubules in living cells.

DQ Ding — 2008-04-10T12:26:54-00:00

Journal of cell science, Vol. 111 ( Pt 6) (March 1998), pp. 701-712.

Using a computerized fluorescence microscope system to observe fluorescently stained cellular structures in vivo, we have examined the dynamics of chromosomes and microtubules during the process of meiosis in the fission yeast Schizosaccharomyces pombe. Fission yeast meiotic prophase is characterized by a distinctive type of nuclear movement that is led by telomeres clustered at the spindle-pole body (the centrosome-equivalent structure in fungi): the nucleus oscillates back and forth along the cell axis, moving continuously between the two ends of the cell for some hours prior to the meiotic divisions. To obtain a dynamic view of this oscillatory nuclear movement in meiotic prophase, we visualized microtubules and chromosomes in living cells using jellyfish green fluorescent protein fused with alpha-tubulin and a DNA-specific fluorescent dye, Hoechst 33342, respectively. Continuous observation of chromosomes and microtubules in these cells demonstrated that the oscillatory nuclear movement is mediated by dynamic reorganization of astral microtubules originating from the spindle-pole body. During each half-oscillatory period, the microtubules extending rearward from the leading edge of the nucleus elongate to drive the nucleus to one end of the cell. When the nucleus reversed direction, its motion during the second half of the oscillation was not driven by the same microtubules that drove its motion during the first half, but rather by newly assembled microtubules. Reversible inhibition of nuclear movement by an inhibitor of microtubule polymerization, thiabendazole, confirmed the involvement of astral microtubules in oscillatory nuclear movement. The speed of the movement fluctuated within a range 0 to 15 micron/minute, with an average of about 5 microm/minute. We propose a model in which the oscillatory nuclear movement is mediated by dynamic instability and selective stabilization of astral microtubules.

Elastic and damping forces generated by confined arrays of dynamic microtubules.

J Howard — 2007-06-19T14:19:15-00:00

Physical Biology, Vol. 3, No. 1. (March 2006), pp. 54-66.

In addition to serving as structural elements and as tracks for motor proteins, microtubules use chemical energy derived from the hydrolysis of GTP to generate forces when growing and shrinking. These forces are used to push or pull on organelles such as chromosomes and the mitotic spindle. If an array of microtubules grows out from a nucleation site and is confined by the periphery of the cell, pushing and pulling forces can give rise to interesting collective phenomena. In this paper, I show that pushing forces center the array provided that the microtubules are dynamic in the sense that they switch from pushing to shrinking after reaching the periphery. Microtubule dynamics of free ends is neither necessary nor sufficient for centering. Buckling can augment the centering force. For small displacements and velocities, the array can be modeled very simply as a damped spring. The dynamic stiffness of the array is orders of magnitude smaller than its static stiffness, and the relaxation time is on the order of the time that it takes for a microtubule to grow from the center to the periphery. Replacement of a dynamic polymer array with an equivalent mechanical circuit provides a bridge between molecular and cellular mechanics.

Microtubule-driven nuclear movements and linear elements as meiosis-specific characteristics of the fission yeasts Schizosaccharomyces versatilis and Schizosaccharomyces pombe.

A Svoboda — 2008-04-10T12:24:37-00:00

Chromosoma, Vol. 104, No. 3. (November 1995), pp. 203-214.

Meiotic prophase in Schizosaccharomyces pombe is characterized by striking nuclear movements and the formation of linear elements along chromosomes instead of tripartite synaptonemal complexes. We analysed the organization of nuclei and microtubules in cells of fission yeasts undergoing sexual differentiation. S. japonicus var. versatilis and S. pombe cells were studied in parallel, taking advantage of the better cytology in S. versatilis. During conjugation, microtubules were directed towards the mating projection. These microtubules seem to lead the haploid nuclei together in the zygote by interaction with the spindle pole bodies at the nuclear periphery. After karyogamy, arrays of microtubules emanating from the spindle pole body of the diploid nucleus extended to both cell poles. The same differentiated microtubule configuration was elaborated upon induction of azygotic meiosis in S. pombe. The cyclic movements of the elongated nuclei between the cell poles is reflected by a dynamic and coordinated shortening and lengthening of the two microtubule arrays. When the nucleus was at a cell end, one array was short while the other bridged the whole cell length. Experiments with inhibitors showed that microtubules are required for karyogamy and for the elongated shape and movement of nuclei during meiotic prophase. In both fission yeasts the SPBs and nucleoli are at the leading ends of the moving nuclei. Astral and cytoplasmic microtubules were also prominent during meiotic divisions and sporulation. We further show that in S. versatilis the linear elements formed during meiotic prophase are similar to those in S. pombe. Tripartite synaptonemal complexes were never detected. Taken together, these findings suggest that S. pombe and S. versatilis share basic characteristics in the organization of microtubules and the structure and behaviour of nuclei during their meiotic cell cycle. The prominent differentiations of microtubules and nuclei may be involved in the pairing, recombination, and segregation of meiotic chromosomes.